Heat Shock Protein 90 Modulates the Unfolded Protein Response by Stabilizing IRE1{alpha}

The molecular chaperone HSP90 regulates stability and functionof multiple protein kinases. The HSP90-binding drug geldanamycininterferes with this activity and promotes proteasome-dependentdegradation of most HSP90 client proteins. Geldanamycin alsobinds to GRP94, the HSP90 paralog located in the endoplasmicreticulum (ER). Because two of three ER stress sensors are transmembranekinases, namely IRE1 ${alpha}$ and PERK, we investigated whether HSP90is necessary for the stability and function of these proteins.We found that HSP90 associates with the cytoplasmic domainsof both kinases. Both geldanamycin and the HSP90-specific inhibitor,514, led to the dissociation of HSP90 from the kinases and aconcomitant turnover of newly synthesized and existing poolsof these proteins, demonstrating that the continued associationof HSP90 with the kinases was required to maintain their stability.Further, the previously reported ability of geldanamycin tostimulate ER stress-dependent transcription apparently dependson its interaction with GRP94, not HSP90, since geldanamycinbut not 514 led to up-regulation of BiP. However, this effectis eventually superseded by HSP90-dependent destabilizationof unfolded protein response signaling. These data establisha role for HSP90 in the cellular transcriptional response toER stress and demonstrate that chaperone systems on both sidesof the ER membrane serve to integrate this signal transductioncascade.

INTRODUCTION

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Introduction

The endoplasmic reticulum (ER) is the primary organelle withinthe cell in which secreted and transmembrane proteins are synthesizedand modified to attain their proper tertiary structure. Variousenvironmental stresses (e.g., glucose deprivation, disturbancein intracellular Ca²⁺ stores, and inhibition of protein glycosylation)lead to the accumulation of incorrectly folded proteins in theER lumen (18). Further, the pathobiology of some diseases hasbeen linked to the deleterious effects of accumulated mutantor misfolded proteins in the ER (4, 14). In eukaryotic cells,the response to unfolded protein accumulation in the ER (termedthe unfolded protein response or UPR) involves three distinctaspects (reviewed in references 15 and 21): (i) translationalattenuation, which reduces the burden of newly synthesized proteinsto be folded by the ER; (ii) transcriptional induction of ERresident molecular chaperones and related stress response proteins,including BiP/GRP78 and GRP94; and (iii) removal of misfoldedproteins from the ER by retrograde transport coupled to theirdegradation by 26S proteasomes at or near the cytoplasmic faceof the ER membrane.

The UPR signal transduction pathway was first elucidated forSaccharomyces cerevisiae. In yeast, an ER-resident transmembraneprotein kinase, Ire1p, is the sole proximal sensor of ER stressleading to activation of the UPR (8, 23, 29). The N terminusof Ire1p senses the perturbed environment within the ER lumen,resulting in oligomerization and trans phosphorylation of itscytoplasmically located kinase domain. This in turn is thoughtto activate an adjacent endoribonuclease activity in the C terminusof Ire1p that excises a translation-inhibitory intron from themRNA encoding the transcription factor Hac1. When Hac1 is efficientlytranslated, it binds to the unfolded protein response elementin the promoters of ER chaperones and other UPR targets andup-regulates their transcription (5, 16, 22, 24).

The mammalian UPR is more complex. In mammals, the ER containstwo Ire1p homologues, the ubiquitously expressed IRE1 ${alpha}$ (38-40)and IRE1ß, which is expressed primarily in gut epithelium(40). In addition, a third resident transmembrane ER kinase,PERK, which is related to the yeast cytosolic Gcn2 kinase andto the mammalian PKR kinase, phosphorylates eIF-2 ${alpha}$ , thereby mediatingtranslational repression in response to UPR activation (11,34). Surprisingly, BiP induction by ER stress is not impairedeither in IRE1 ${alpha}$ ^-/- IRE1ß^-/- double knockout cells obtainedfrom mouse embryos (39) or in murine embryonic stem cells inwhich the IRE1 ${alpha}$ gene was inactivated by gene disruption (19),suggesting the presence of IRE-independent or -compensatingpathways in mammalian cells that regulate ER stress-inducedtranscriptional responses.

The ER resident transmembrane transcription factor ATF6 is anadditional transactivator of the UPR. The cytosolic portionof this protein encodes a transcriptional activation domainthat, when cleaved from the membrane in response to ER stress,migrates to the nucleus, where it is able to up-regulate theBiP gene and other stress-responsive genes (12, 20, 41, 45).Whether ATF6 and IRE1 function as serial components of a singlepathway or instead represent parallel but overlapping pathwaysremains under investigation.

A number of key regulatory kinases, including the soluble serine/threoninekinases Raf1 (32, 36, 42), Akt (31, 35), Gcn2 (10), PKR (9),and the type I transmembrane tyrosine kinase p185/ErbB2 (6,43), depend on interaction with the molecular chaperone HSP90for stability and function. Where investigated, HSP90 has beenfound to associate with the kinase domains of these proteins,and interference with this association by the HSP90 bindingdrug geldanamycin (GA) results in their rapid destabilizationand proteasome-mediated degradation (for a review, see reference25). HSP90 has not previously been implicated in the ER stressresponse. However, because both PERK and IRE1 are type I transmembranekinases, and given the HSP90 dependence of both Gcn2 and PKR,we decided to investigate the possibility that HSP90 may participatein the mammalian UPR by regulating the stability and functionof IRE1 and/or PERK. In addition, we sought to determine whetherthe recently reported ability of GA to induce only a transientUPR (17) was due to disruption of HSP90-mediated effects onthe UPR signaling pathway.

Here we show that HSP90 does indeed associate with the cytoplasmicdomains of both ER transmembrane kinases and that its continuedassociation is required for the stability of these proteins.Finally, our data show that the ability of GA to transientlyinduce the UPR depends not on its binding to HSP90 but on itsinteraction with GRP94, the ER-resident paralog of HSP90. Thiseffect is time limited because of the GA-induced loss of HSP90-dependentIRE1 activity.

MATERIALS AND METHODS

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Materials and Methods

Cell culture and transient transfections. NIH 3T3 fibroblasts and HEK 293 and COS7 cells were grown inDulbecco's modified Eagle's medium containing 10% fetal bovineserum, 2 mM L-glutamine, and 1% Fungizone (BioWhittaker, Walkersville,Md.). AR42J rat pancreatic tumor cells were maintained in F12Kmedium (American Type Culture Collection, Manassas, Va.) supplementedwith 15% fetal bovine serum, 2 mM L-glutamine, and 1% Fungizone.

For transient-transfection studies, HEK 293 and COS7 cells weregrown in 6-cm-diameter dishes. Cells were transfected with 0.5to 1 µg each of the Flag-tagged cDNAs encoding eitherfull-length human IRE1 ${alpha}$ (amino acids 1 to 1115) or ${Delta}$ IRE1 (lackingthe cytoplasmic domain; amino acids 725 to 1115), using Lipofectamine-Plus(Gibco BRL, Rockville, Md.) according to the manufacturer'sinstructions. When necessary, 24 h after transfection, cellswere treated with GA (according to the experiment) and thenlysed with cold TNESV buffer (50 mM TRIS-HCl [pH 7.4], 1% NonidetP-40, 2 mM EDTA, 100 mM NaCl, 1 mM sodium orthovanadate) containingprotease inhibitors. The full-length IRE1 ${alpha}$ and ${Delta}$ IRE1 cDNAs, clonedin pcDNA3.1 plasmid, were a kind gift from K. Imaizumi (OsakaUniversity, Osaka, Japan) and have been described previously(13).

The ATF6 expression plasmid, pCGN-ATF6 (containing a hemagglutinin[HA] tag at the N terminus), was obtained from R. Prywes (ColumbiaUniversity, New York, N.Y.), and has been previously described(41). COS7 cells were transiently transfected as described above.At 20 to 24 h after transfection, cells were treated with GA,the GA derivative 514, or tunicamycin, according to experimentalconditions. Both cleaved and uncleaved forms of transientlyexpressed HA-tagged ATF6 protein were detected in cell lysatesby sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting as described previously (43).

Antibodies and immunoprecipitations. HSP90 antibodies, SPA-830 and SPA-835, were purchased from StressGenBiotechnologies Corp. (Victoria, British Columbia, Canada).HA, BiP, and XBP1 antibodies were purchased from Santa CruzBiotechnologies (Santa Cruz, Calif.). IRE1 and PERK polyclonalantisera were obtained from D. Ron (New York University, NewYork, N.Y.). Anti-FLAG-M2 antibody was purchased from UpstateBiotechnology (Lake Placid, N.Y.). Immunoprecipitation assayswere performed as described previously (43). Briefly, solubleproteins (400 µg for each condition) were immunoprecipitatedwith 1 to 3 µg of anti-Flag, anti-PERK, or anti-IRE1 antibodies,and immune complexes were bound on protein A-Sepharose beadsfor 6 h at 4°C (with rotation). The beads were washed fourtimes with TNESV buffer, and bound proteins were resolved bySDS-7.5 or 10% PAGE and transferred to nitrocellulose membranesfor Western blot analysis.

Stress inducers. GA and its HSP90-preferring derivative 514 (NSC#658514) wereobtained from the Developmental Therapeutics Program (NationalCancer Institute), dissolved in dimethyl sulfoxide at stockconcentrations of 2 and 5 mM, respectively, and diluted to theirfinal concentrations using aqueous media. Tunicamycin, dithiothreitol(DTT), and thapsigargin were purchased from Sigma (St. Louis,Mo.) and dissolved in dimethyl sulfoxide or water at stock concentrationsof 2.5 mg/ml, 1 M, and 1 mM, respectively.

GST-HSP90 binding of IRE1. Equal amounts of protein lysate from transfected cells or fromcells containing high levels of endogenous IRE1 (AR42J) wereincubated with glutathione Sepharose 4B beads (Amersham Pharmacia,Uppsala, Sweden) that had been previously loaded with purifiedglutathione S-transferase (GST)-HSP90 (a gift of D. Toft, MayoClinic, Rochester, Minn.). The samples were rotated at 4°Cfor 2 h and then washed thoroughly with TNESV buffer. Washedbeads were boiled in protein loading buffer and analyzed bySDS-PAGE. Western blotting was performed with either anti-Flagor anti-IRE 1 antibodies.

Determination of IRE1 half-life in the presence or absence of GA. COS7 cells were transfected with IRE1 plasmids as describedabove. These cells or AR42J cells (expressing abundant endogenousIRE1) were treated with GA for 8 h or as indicated. The cellswere then starved for 30 min in methionine/cysteine-free mediaand pulse-labeled with 100 µCi of [³⁵S]methionine/cysteine(Tran ³⁵S-label; ICN, Costa Mesa, Calif.)/ml for 60 min. Thecells were washed twice and further incubated in complete Dulbecco'smodified Eagle's medium supplemented with 10% fetal calf serumin the presence or absence of GA (1 µM). At various timesafter being returned to complete medium, cells were lysed andIRE1 proteins were immunoprecipitated with either anti-Flagantibody or anti-IRE1 antisera. Samples were analyzed by SDS-PAGEand autoradiography. Band densities were quantified by NIH Imagesoftware using a Macintosh 9500 computer.

RESULTS

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Results

Association of HSP90 with IRE1. Plasmids expressing Flag-tagged full-length IRE1 ${alpha}$ or IRE1 ${alpha}$ lackingthe cytoplasmic kinase and RNase domains ( ${Delta}$ IRE1) were transfectedinto COS7 cells. Cells were lysed 24 h later, and protein lysateswere subjected to immunoprecipitation using an anti-Flag antibody.After SDS-PAGE and electrotransfer to nitrocellulose, blotswere probed with an antibody to HSP90 (Fig. 1A). HSP90 was clearlycoprecipitated with full-length IRE1 but not with ${Delta}$ IRE1, eventhough ${Delta}$ IRE1 was reproducibly expressed to a greater degree thanwas wild-type IRE1. By transfecting 1/10 the amount of ${Delta}$ IRE1as wild-type IRE1, approximately equivalent steady-state levels(Fig. 1A, bottom panel) were obtained. In order to rule outthe possibility that association of HSP90 with IRE1 was a transfectionartifact, we immunoprecipitated endogenous IRE1 ${alpha}$ from AR42J,a pancreatic cell line with a well-developed ER. HSP90 readilycoprecipitated with IRE1 from untreated cells (Fig. 1B), whilea brief exposure (3 h) of AR42J cells to GA disrupted IRE1-HSP90association (Fig. 1B) without altering the steady-state levelsof either IRE1 or HSP90 (Fig. 1B, right panels). We recentlyidentified a chemically similar benzoquinone ansamycin, heredesignated 514, that has a 90-fold better binding affinity forHSP90 than for the chaperone's ER paralog, GRP94 (43). Usingthis compound, we also observed disruption of association ofIRE1 with HSP90 (Fig. 1B). Last, we used purified GST-HSP90protein to pull down both tagged IRE1 exogenously expressedin HEK293 cells and endogenous IRE1 in AR42J cells (Fig. 1C and D).Consistent with the in vivo binding data, GST-HSP90was unable to pull down equivalent amounts of ${Delta}$ IRE1 transientlyexpressed in HEK293 cells (Fig. 1C). Taken together, these datasupport the hypothesis that full-length IRE1 associates withHSP90 via IRE1's cytoplasmic domain and that GA binding to HSP90disrupts this association.

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FIG. 1. HSP90 associates with IRE1 but not with cytoplasmic domain-deleted IRE1. Cos7 cells were transfected with IRE1-FLAG or ${Delta}$ IRE1-FLAG plasmids and lysed in TNESV after 24 h. Untransfected AR42J cells were treated or with GA or its derivative 514 (1 µM) of left untreated (lane 0) for 3 h, and then the cells were lysed similarly. Soluble proteins (400 µg) were immunoprecipitated with either 3 µg of anti-Flag antibody (A) or IRE1 antibody (B), and complexes were bound on protein A-Sepharose beads for 2 h at 4°C. The beads were washed four times with TNESV, bound proteins were resolved by SDS-10% PAGE and transferred to nitrocellulose membranes. Western analysis was performed with either anti-HSP90 or anti-IRE antibodies. Expression levels of transiently transfected wild-type IRE1 and ${Delta}$ IRE1 are shown for comparison in panel A, and HSP90 and IRE1 steady-state levels in cell lysates are shown for comparison in panel B. HEK293 cells were transfected with IRE1-FLAG or ${Delta}$ IRE1-FLAG plasmids. After 24 h, the cells were lysed and 1 mg of total proteins from each condition was subjected to GST-HSP90 binding in the presence of 400 µl of glutathione-Sepharose 4B, which had been previously saturated with GST-HSP90 (C). The binding of native IRE1 (in AR42J lysates) to either GST-HSP90 or GST alone is shown in panel D.

Disruption of HSP90 association coincides with loss of IRE1 protein. Both COS7 cells transiently transfected with either full-lengthIRE1 or ${Delta}$ IRE1 and AR42J cells expressing endogenous IRE1 weretreated with increasing concentrations of GA for 24 h. Steady-statelevels of Flag-tagged IRE1 (COS7 cells) or endogenous IRE1 (AR42J)proteins were monitored by Western blotting. The data in Fig.2 show dose- and time-dependent depletion of both transfectedand endogenous IRE1, while ${Delta}$ IRE1 remained completely resistantto GA. Thus, GA sensitivity of IRE1 paralleled HSP90 binding,since ${Delta}$ IRE1 did not bind HSP90 and was not sensitive to GA. Theseresults are identical to those obtained with another type Itransmembrane kinase, ErbB2 (43).

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FIG. 2. Geldanamycin destabilizes IRE1 but not cytoplasmic domain-deleted IRE1. Cos7 cells were transfected with full-length IRE1-FLAG or ${Delta}$ IRE1-FLAG. Cos7 cells (A) or AR42J cells (expressing endogenous IRE1) (B) were incubated for 24 h with increasing concentrations of GA. (C) AR42J cells were exposed to 1 µM GA for the time periods shown. Cells were lysed, and equivalent amounts of protein were analyzed by SDS-PAGE and Western blotting with Flag antibody (A) or IRE1 antibody (B and C). In panels B and C, tubulin was blotted to demonstrate equal loading of protein.

GA reduces the half-life of IRE1 protein. Because GA did not affect IRE1 mRNA levels (data not shown),we expected that its effect on IRE1 protein was posttranslational.Thus, we examined drug effects on the IRE1 protein half-life.COS7 cells transiently transfected with full-length IRE1 andAR42J cells expressing the endogenous protein were pulse-labeledwith [³⁵S]methionine/cysteine as described in Materials andMethods and then chased for increasing times in nonradioactivecomplete medium. In each case, one set of cells was continuouslyexposed to GA while another set remained untreated. The datashow that synthesis of either exogenously expressed or endogenousIRE1 in the presence of GA resulted in a protein with a markedlyreduced half-life (Fig. 3). In COS7 cells the half-life of Flag-IRE1was reduced from >5 h to <1 h, while in AR42J cells thehalf-life of IRE1 ${alpha}$ was reduced from approximately 3 h to <1h. These results are consistent with the effect of GA on otherHSP90 client proteins (26).

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FIG. 3. GA decreases the half-life of newly synthesized IRE1. Transiently transfected Cos7 (A) and untransfected AR42J (B) cells were treated with GA (1 µM) 12 h after transfection or left untreated. After an additional 8 h, the medium was replaced with methionine/cysteine-free medium for 30 min, and cells were pulse-labeled with 100 µCi of [³⁵S]methionine/cysteine/ml for 60 min. At 1 h, cells were placed in complete, nonradioactive medium (time zero) and chased for the times shown. GA remained in all drug-treated samples for the duration of the experiment. Proteins were immunoprecipitated with either anti-Flag (A) or anti-IRE1 (B) antibodies. Samples were analyzed by SDS-PAGE and autoradiography. Band densities of scanned films were quantified using NIH Image software to create the graphs shown.

Binding of benzoquinone ansamycin to GRP94 induces an ER stress response, while binding to HSP90 eventually abrogates such a response. Since GA depletes IRE1 protein within several hours, we suspectedthat the drug would interfere with the ER's transcriptionalresponse to stress-inducing stimuli. On the other hand, Lawsonet al. had previously shown that GA itself induces an ER stressresponse, although unlike agents such as thapsigargin or tunicamycin,the GA-induced stress response was relatively short-lived (17).In the present experiments, we first confirmed that the levelof IRE1 protein was significantly reduced in cells exposed toeither GA or 514 for 12 h (Fig. 4A). Concomitantly, exposureto either HSP90 inhibitor for 12 h abrogated the induction ofBiP protein synthesis following a 4-h challenge with tunicamycin(TM) (Fig. 4B). Inhibition of the response to TM was not dueto inhibition of general protein synthesis in cells exposedto either GA or 514 for 12 h. Thus, untreated cells pulsed brieflywith radiolabeled methionine incorporated 1.77 x 10⁶ cpm/mgof protein (trichloroacetic acid-precipitable counts), whilecells exposed to GA or 514 for 12 h incorporated 1.78 x 10⁶and 1.87 x 10⁶ cpm/mg of protein, respectively. In contrastto the results obtained following prolonged exposure to HSP90inhibitors, a 4-h exposure to GA itself produced a robust BiPresponse, equivalent to that caused by TM (Fig. 4C). However,4-h challenge with 514 caused only a negligible BiP response(Fig. 4C). Thus, although both GA and 514 blocked the stressresponse after prolonged exposure, after a brief exposure, onlyGA was able to promote a stress response.

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FIG. 4. Both GA and 514, a GA derivative that has a 2-log-greater affinity for HSP90 than for GRP94, inhibit the UPR after prolonged exposure, but only GA induces the UPR after short exposure. (A) AR42J cells (which express high levels of IRE1) were exposed to either GA (1 µM) or 514 (1 or 10 µM) for 12 h. IRE1 and tubulin proteins were detected in whole-cell lysates by Western blotting. (B) AR42J cells were pretreated with either the GA derivative 514 or GA for 12 h before the stress-inducer TM was added (for an additional 4 h). BiP protein was detected by [³⁵S]methionine pulse-labeling for 30 min, and tubulin protein was detected by Western blotting. (C) AR42J cells were exposed to TM, GA, or 514 for 4 h and processed for BiP and tubulin protein detection as was done for panel A. (D) Cos7 cells were transfected with pCGN-ATF6, an ATF6 expression plasmid containing a HA epitope tag. Twenty hours after transfection, cells were either left untreated (lanes 1 and 7), treated with TM for 4 h (lane 2), pretreated with GA for 12 h, followed by TM for an additional 4 h (lane 3), pretreated with 514 for 12 h, followed by TM for 4 h (lane 4), treated with only GA for 12 h (lane 5), or treated with only 514 for 12 h (lane 6). In order to better detect the ATF6 cleavage product induced by ER stress, we treated all cells with the proteasome inhibitor PS-341 (0.5 µM) during exposure to the ER stress agent. In lanes 1, 5, 6, and 7, PS-341 was added for the final 4 h of incubation prior to lysis. Following cell lysis, equivalent amounts of soluble proteins were separated by SDS-PAGE and analyzed for stress-induced ATF6 cleavage by Western blotting using an anti-HA antibody.

GA inhibits ER stress-induced cleavage and activation of ATF6. IRE1 is not the sole mediator of the transcriptional componentof the UPR. Indeed, the BiP response to ER stress remains essentiallyintact in IRE1^-/- cells (19). Thus, the dramatic inhibitionof ER stress-mediated BiP induction by prolonged inhibitionof HSP90 requires additional explanation. ATF6, an ER transmembranelocalized transcription factor, has also been implicated inthe transcriptional response to ER stress, since its cytoplasmicdomain can activate BiP transcription upon cleavage from themembrane (12, 20, 41, 45). Since ATF6 cleavage is necessaryfor its transcriptional activity, we transfected cells withan ATF6 expression plasmid, and 20 h later we determined theeffects of GA and 514 on cleavage of ATF6 in response to TM.A proteasome inhibitor was included in these incubations, sincethe cleaved fragment of ATF6 is very unstable but can be protectedby proteasome inhibition (44). Although exogenous overexpressionof ATF6 can lead to spontaneous cleavage, we successfully titeredthe amount of plasmid transfected to avoid this problem (Fig.4D, lanes 1 and 7). While TM at 4 h caused easily detectablecleavage of ATF6 (Fig. 4D, compare lanes 1 and 2), prior exposureto both HSP90 inhibitors for 12 h resulted in much less ATF6cleavage following TM challenge (Fig. 4D, compare lane 2 withlanes 3 and 4). The HSP90 inhibitors alone caused minimal ATF6cleavage (Fig. 4D, lanes 5 and 6). Thus, prevention of stress-inducedATF6 cleavage is an additional means by which HSP90 inhibitionaffects the transcriptional arm of the UPR.

GA promotes BiP dissociation from IRE1, but other stressors do not disrupt the HSP90-IRE1 interaction. Agents that induce ER stress have been shown to disrupt associationof the ER chaperone BiP with the lumenal domain of IRE1 (2,28). We wished to determine whether GA had a similar activityand at the same time determine if ER stress generally resultsin dissociation of HSP90 from the cytoplasmic domain of IRE1.Therefore, we exposed AR42J cells to DTT for 30 min, to TM for2 h, or to GA for 1 or 2 h. After immunoprecipitation of endogenousIRE1, coprecipitation of BiP was examined by Western blotting.We found that each of the three agents efficiently disruptedBiP-IRE1 association when present for the times shown (Fig.5A). In contrast, and unlike GA, neither TM nor DTT dissociatedHSP90 from IRE1 (Fig. 5B). Specificity of the HSP90 coimmunoprecipitationwith anti-IRE antibody was confirmed by the inability of anti-FLAGantibody to coimmunoprecipitate HSP90 (Fig. 5B, bottom panel,and Fig. 1A). Thus, although GA mimics the ability of otherER stress agents in disrupting BiP/IRE1 complexes, disruptionof HSP90-IRE1 association is neither a general feature nor aprerequisite of the ER stress response.

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FIG. 5. ER stressors, including GA, dissociate IRE1 from BiP but do not dissociate HSP90 from IRE1. (A) AR42J cells were treated with GA (2 µM) for 1 or 2 h, TM (2.5 µg/ml) for 2 h, or DTT (10 mM) for 30 min. After lysis, 400 µg of soluble proteins were immunoprecipitated with 2 µg of IRE1 antibody, and the complexes were bound on protein A-Sepharose beads. Bound proteins were resolved by SDS-7.5% PAGE and transferred to nitrocellulose membranes. Western analysis was performed with anti-IRE1 and anti-BiP antibodies. (B) In a similar experiment, HSP90 coprecipitation following anti-IRE1 immunoprecipitation was examined (by an HSP90 Western blot) in cells treated with TM or DTT. The specificity of HSP90 coprecipitation with IRE1 is shown by lack of an HSP90 signal in anti-FLAG immunoprecipitates.

The ER transmembrane kinase PERK also associates with HSP90 and is destabilized by GA. We next examined whether the other ER stress-activated kinasePERK was also an HSP90 client protein. As shown in Fig. 6A andB, HSP90 does coprecipitate with PERK in AR42J cells. Briefexposure to GA disrupts this association, and prolonged exposureto GA results in a decline in PERK protein level (due to a decreasedprotein half-life; data not shown).

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FIG. 6. The ER transmembrane kinase PERK associates with HSP90 but is functionally less sensitive to GA than is IRE1. (A) AR42J cells were treated with GA (1 µM) for the times indicated, cells were lysed, and proteins were resolved by SDS- 10% PAGE and transferred to nitrocellulose membranes. Western analysis of PERK steady-state levels was performed with an anti-PERK antibody. Tubulin was blotted to demonstrate equal loading of protein. (B) AR42J cells were left untreated or treated with GA (1 µM for 4 h) and then lysed, and 400 µg of soluble proteins were immunoprecipitated with 2 µg of anti-PERK antibody. Antibody complexes were bound on protein A-Sepharose beads, washed, eluted, and resolved by SDS-10% PAGE. After transfer to a nitrocellulose membrane, Western analysis of coprecipitated HSP90 was performed. The immunoprecipitates were also blotted for PERK to demonstrate its equivalent pulldown in the presence and absence of GA. (C) AR42J cells were treated with GA (1 µM) for the indicated times. The cells were then pulse-labeled with 100 µCi of [³⁵S]methionine/cysteine/ml for 30 min and lysed. Equal amounts of total soluble proteins were analyzed by SDS-PAGE and autoradiography. (D) AR42J cells were treated with thapsigargin (Th; 0.5 µM) for 0.5 h (filled diamond), GA (1 µM) for 0.5, 1, 2, or 24 h (filled circles) or with a combination of GA and Th (filled square and filled triangle; GA was added for the indicated time, followed by Th for 30 min). Cells were then pulse-labeled and processed as for panel C. Equal amounts of total cell proteins were analyzed by SDS-PAGE and autoradiography.

GA fails to block thapsigargin-induced translational inhibition. PERK activation leads to translational attenuation (11, 34).In order to determine whether PERK function requires PERK-HSP90association, we tested whether GA can block stress-induced translationalinhibition. Using a radioactive methionine pulse to measuretranslation, we observed that GA itself caused a noticeablebut transient attenuation of translation (Fig. 6C). However,preexposure of cells to GA for 1 or 24 h did not prevent thetranslational attenuation observed following a 30-min exposureto thapsigargin (Fig. 6D). These data show that although PERKstability is reduced when it is disassociated from HSP90, resultingin a slow decline in its steady-state level, the activity ofthe remaining PERK protein is sufficient to inhibit proteinsynthesis during activation of the ER stress response.

DISCUSSION

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Discussion

Our data demonstrate the involvement of HSP90 in the transcriptionalarm of the UPR. HSP90 associates with both exogenously introducedand endogenously expressed IRE1 ${alpha}$ , and the HSP90 inhibitor GApromotes dissociation of the chaperone from IRE1 concomitantwith a marked reduction in the half-life of the kinase. SinceIRE1 lacking its cytosolic domain neither associates with HSP90nor is sensitive to GA, it is likely that drug sensitivity isdependent on chaperone interaction with the kinase domain ofIRE1, as is the case with other HSP90 client transmembrane kinases.

In accord with the dependence of IRE1 ${alpha}$ stability on HSP90 binding,prolonged exposure of cells to GA inhibits BiP induction causedby ER stress. However, in agreement with and extension of thestudy of Lawson et al. (17), short-term exposure to GA mimicsthe activity of other ER stressors in that it stimulates BiPprotein synthesis and promotes the dissociation of IRE1 ${alpha}$ fromBiP. However, the GA-induced disassociation of HSP90 from IRE1is not a prerequisite for IRE1 activation, since other stressagents, including DTT and Tm, do not share this property withGA.

How can GA both induce and inhibit ER stress-dependent transcription,depending on the length of exposure? Our present data offeran explanation for the transience of the GA-induced UPR (17).GA interacts with similar affinities with both HSP90 and itsER paralog, GRP94 (6, 43). If the binding of GA to GRP94 interferedwith this chaperone's role in protein maturation, thereby increasingthe requirement for BiP to bind to "stalled" incompletely foldedproteins transiting the ER, the resultant release of BiP fromthe luminal domain of IRE1 would induce the ER stress response(2). Indeed, GA is reported to interfere with the maturationand secretion of at least two proteins, bile-salt-dependentlipase (27) and immunoglobulin lambda light chain (1), thatboth interact with GRP94 during their transit through the ER.A third protein, the toll receptor, also depends on GRP94 formaturation as it transits the ER (30); however, possible effectsof GA on the toll receptor have not yet been examined. At thesame time, the deleterious effect of GA on IRE1 should eventuallyinterfere with the cell's transcriptional response to ER stress.The combined but opposing effects of GA on both HSP90 and GRP94could explain the transitory stress response induced by thisdrug. This proposed mechanism is supported by the data obtainedwith the GA derivative 514. Unlike GA, 514 binds to GRP94 witha K_d(GRP94) of 90 µM, compared to a K_d(HSP90) of 1 µMfor HSP90 (43). Since 514 proved to be a poor inducer of BiPsynthesis upon short exposure but an efficient inhibitor ofBiP response to TM upon prolonged treatment, the data are consistentwith the hypothesis that the former event depends on bindingof drug to GRP94 while the later process requires binding ofdrug to HSP90.

Given that BiP responds to ER stress in cells obtained fromIRE1^-/- mice, it is unlikely that the ability of GA to significantlyimpair BiP induction is due solely to its effect on IRE1. Thetranscription factor ATF6 may represent a bifurcation of thetranscriptional arm of the UPR in mammalian cells. Recent datafrom several laboratories suggest that IRE- and ATF6-mediatedsignaling pathways converge to maximally induce the UPR transcriptionalresponse (3, 7, 19, 46). In response to ER stress, ATF6 migratesfrom ER to Golgi, where it is cleaved from a 90-kDa membrane-associatedform to an approximately 50-kDa soluble active fragment (7),which migrates to the nucleus and activates transcription. Althoughwe were unable to detect significant GA- or 514-induced proteolyticcleavage of transfected ATF6, pretreatment with both HSP90 inhibitorsmarkedly reduced the ability of TM to cause ATF6 cleavage. Thus,by demonstrating that GA can interfere with both transcriptionalarms (IRE1 and ATF6) of the UPR, these data offer an explanationas to how GA can so dramatically impair the BiP response toER stress. However, complicating interpretation of these findings,GA did not affect the steady-state level of unprocessed ATF6,nor did we observe HSP90 in association with either form ofATF6 (data not shown). Also, BiP has been reported to bind tothe luminal domain of ATF6, much as it does to IRE1 and PERK,and removal of BiP binding sites results in constitutive translocationof ATF6 to the Golgi, where it is cleaved (33). Although basedon this paradigm we would have expected GA to activate ATF6,it is possible that the GA effect on ATF6 is indirect and thatthe drug interferes elsewhere in the ATF6 cleavage pathway toinhibit processing of the transcription factor. One possibilitycurrently being explored is that the inhibition by GA of stress-inducedATF6 activation is dependent on drug activity toward one ormore upstream HSP90 client kinases whose phosphorylation ofATF6 is a prerequisite for its cleavage (or transport from ERto Golgi). Indeed, phosphorylation of ATF6 by p38 mitogen-activatedprotein kinase has been shown to modulate its transcriptionalactivity in the cellular context of cardiac myocytes (37). Whetherp38 kinase is activated by ER stress and is sensitive to GA,and whether ATF6 phosphorylation is a necessary signal for eitherits transit to the Golgi or its proteolytic cleavage, are currentlyunder investigation.

PERK, a mediator of ER stress-induced translational inhibition,is a second ER transmembrane kinase found in mammalian cells.We show here that PERK also associates with HSP90 and that GAdisrupts this association, slowly reducing the steady-statelevel of the protein over many hours. However, unlike IRE1 ${alpha}$ ,PERK activity is much less dependent on HSP90. Thus, even long-termexposure to GA, although it significantly reduces the steady-statelevel of the PERK protein, does not prevent an ER stressor fromstimulating PERK autophosphorylation (data not shown) or PERK-mediatedtranslational inhibition. These data suggest that PERK levelsare in excess of what is necessary for its activity to be manifest.The lack of sensitivity of PERK to both HSP90 and GRP94 inhibitionby GA is somewhat surprising given the apparent interchangeabilityof the luminal domains of PERK and IRE (2). In addition, twocytosolic kinases related to PERK, Gcn2 in yeast and PKR inmammals, both depend on HSP90 for their activity and stability(9, 10). Further examination of the nature of PERK-HSP90 andPERK-BiP interaction in AR42J and in other cell types may ultimatelyexplain these discrepancies.

An interesting possibility suggested by our data is that allproximal sensors of ER stress are not equally sensitive to anincoming signal. Disruption of GRP94 function by GA may causeonly a limited increase in protein load in the ER and thus maybe recognized as a minor stress, not a strong enough stimulusto warrant shutting down general protein translation or activatingATF6 but significant enough to moderately stimulate transcriptionof ER chaperones to compensate. The IRE1 ${alpha}$ signaling pathway maythus represent the cellular response to a low level of ER stress.In this model, activation of PERK and/or ATF6 would occur onlywhen the stress is prolonged or severe enough to require morethan a moderate increase in the level of ER chaperones. Sucha graded response model, recently proposed by several groups(19, 46), would help to explain why, when compared to yeast,the regulation of the UPR has become so complex in mammaliancells.

In summary, our data demonstrate that HSP90 is an importantcomponent of the transcriptional arm of the UPR, much as thechaperone plays an important role in the cellular response tocytoplasmic stress. However, while nonspecific binding to misfoldedor unfolded proteins is thought to be the primary function ofHSP90 during cytoplasmic stress, its role in the promulgationof the ER stress response depends instead on HSP90's specificassociation with at least one ER resident transmembrane kinase,IRE1 ${alpha}$ .

FOOTNOTES

* Corresponding author. Mailing address: 9610 Medical Ctr. Dr., Suite 300, Rockville, MD 20850. Phone: (301) 402-3128, ext. 318. Fax: (301) 402-4422. E-mail: len{at}helix.nih.gov.

Present address: INSERM, Laboratoire de Genetique Moleculaire,UMR8541 du CNRS, Ecole Normale Superieure, 75005 Paris, France.

REFERENCES

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